Dental Mesenchymal Stem Cell Secretome: An Intriguing Approach for Neuroprotection and Neuroregeneration

Mesenchymal stem cells (MSCs) are known for their beneficial effects and regenerative potential. In particular, dental-derived MSCs have the advantage of easier accessibility and a non-invasive isolation method. Moreover, thanks to their neural crest origin, dental MSCs seem to have a more prominent neuroregenerative potential. Indeed, in basal conditions they also express neuronal markers. However, it is now well known that the beneficial actions of MSCs depend, at least in part, on their secretome, referring to all the bioactive molecules released in the conditioned medium (CM) or in extracellular vesicles (EVs). In this review we focus on the applications of the secretome derived from dental MSCs for neuroregeneration and neuroprotection. The secretomes of different dental MSCs have been tested for their effects for neuroregenerative purposes, and the secretomes of dental pulp stem cells and stem cells from human exfoliated deciduous teeth are the most studied. Both the CM and EVs obtained from dental MSCs showed that they are able to promote neurite outgrowth and neuroprotective effects. Interestingly, dental-derived MSC secretome showed stronger neuroregenerative and neuroprotective effects compared to that obtained from other MSC sources. For these reasons, the secretome obtained from dental MSCs may represent a promising approach for neuroprotective treatments.


Introduction
Mesenchymal stem cells (MSCs) are multipotent cells with great potential for regenerative medicine [1]. MSCs were first isolated in the bone marrow by Friedenstein et al. [2,3]. However, the term MSCs was coined later by Caplan, indicating their multipotent differentiation ability to give rise to mesodermal lineage [4]. In 2006, Dominici et al. established the criteria to classify MSCs, which are the plastic adherence ability in standard culture conditions, the expression of CD105, CD73 and CD90, the lack of CD45, CD34, CD14 or CD11b, CD79alpha or CD19 and HLA-DR surface molecules, and the differentiation potential toward osteoblasts, adipocytes and chondroblasts in vitro [5].
Since the first discovery, MSCs have been isolated from different tissues. Regarding dental tissues, in 2000 Gronthos et al. firstly isolated a population of MSCs from dental pulp cells, with similar properties to bone marrow MSCs (BMSCs) [6]. Since then, different dental-derived cells have been found to possess stem cell properties and were named according to their tissue of origin, including dental pulp stem cells (DPSCs), stem cells from human exfoliated deciduous teeth (SHEDs), periodontal ligament stem cells (PDLSCs), dental follicle stem cells (DFSCs), stem cells from apical papilla (SCAPs), and gingival MSCs (GMSCs) [7].
Dental MSCs have the advantages of being easily accessible with minimally invasive procedures [8], expandable with relative genomic stability for a long period of time, and show immunomodulatory properties [9]. Moreover, they are also able to differentiate toward the mesodermal lineage, but they also show the ability to transdifferentiate into ectodermal and endodermal lineages [10].
Dental MSCs have neural crest origin and for this reason they show more potent neurogenic capabilities compared to other MSCs [11]. Thanks to their origin, dental MSCs express some neural progenitor and mature cell markers, even when not exposed to neural induction medium and in standard culture conditions, such as nestin, β-3 tubulin, neurotrophin receptors, and neurofilaments [12,13]. In addition, dental MSCs show a greater differentiation potential for neurogenesis compared to other MSCs types [14,15]. Thus, dental MSCs, thanks to their differentiation potential and paracrine effects, may represent a good source of MSCs for the treatment of neurodegenerative disorders and for neural regeneration [16][17][18][19][20].
The beneficial properties of MSCs are often associated with their differentiation potential. Indeed, MSCs differentiating toward neuronal cells may replace degenerated ones. However, it is now well accepted that MSCs' regenerative and protective effects are mediated also by their secretome. In this review, we focus on the secretome obtained by dental MSCs, showing its potential for neuroprotection and neuroregeneration in preclinical models.
The application of the secretome for cell-free therapy seems promising and has the advantage of not having the ethical limits related to the use of stem cells, and shows low immunogenicity [22]. In addition, some reports indicate only a limited survival of MSCs after transplantation [23].
EVs may also play central roles in cell-free therapies. EVs are membrane-bound bilayered lipid particles, secreted by different cell types, carrying a cargo of biological molecules from their parent cells. They are important mediators of biological information in intercellular cell signaling from the parent into a recipient cell. EVs are classified as microvesicles (MVs), exosomes (EXOs), and apoptotic bodies on the basis of their size but also of other features such as biogenesis and release pathways [24,25]. MVs are produced through direct budding from the cell plasma membrane. On the contrary, EXOs are smaller and originate by an inward budding of the limiting membrane of early endosomes, which mature into multivesicular bodies during the process. After the fusion with the plasma membrane, multivesicular bodies release EXOs into the extracellular milieu [24,26]. EVs, thanks to their surface molecules, can target the recipient cells. Once attached to a target cell, EVs can promote signaling via receptor-ligand interactions or can be internalized by endocytosis, phagocytosis or fuse with the target cell's membrane and release their content into the cytoplasm [27,28]. EVs released by MSCs contain proteins, lipids, mRNA, microRNA (miRNA), and cytokines. These vesicles release their contents into target cells, modulating their activity and potentially inducing restorative processes [29].

Dental MSCs Secretome
Interestingly, the secretome profile may be influenced by different MSC sources [30]. For this reason, dental MSCs may present differences in secretome composition compared to other MSCs.
The analysis of SCAPs secretome has evidenced a total of 2046 proteins including chemokines, angiogenic, immunomodulatory, antiapoptotic, and neuroprotective factors other than extracellular matrix (ECM) proteins. Interestingly, the levels of 151 proteins were different by at least twofold compared to BMSCs. Indeed, SCAPs showed increased levels of proteins involved in metabolic processes, transcription, chemokines and neurotrophins while they presented a reduction of those associated with biological adhesion, developmental processes, immune function, ECM proteins and proangiogenic factors [31]. regulation, cellular processes, metabolic processes, binding, and catalytic activity. Specifically, the target genes of the upregulated piRNAs in SCAP-EXOs were enriched in the mitogen-activated protein kinase (MAPK) signaling pathway, Ras signaling pathway, and citrate cycle signaling pathway. On the contrary, the target genes of downregulated piR-NAs in SCAP-EXOs were enriched in the p53 signaling pathway and Epstein-Barr virus infection signaling pathway [43].
It is important to notice that donor age and microenvironmental conditions in vitro may also influence secretome composition. DPSC-CM obtained in normoxic conditions was reported to be enriched in molecules with anti-inflammatory, tissue repair, and regenerative properties compared to CM obtained in hypoxic conditions [44]. Moreover, secretome collected from 5% O 2 cultured DPSCs showed higher stimulatory effects on proliferation and migration of mouse embryonic fibroblast NIH3T3 cells and on neuronal differentiation of SH-SY5Y cells [45]. The quantity and size of EXOs and their tetraspanins expression may vary dependent on the medium used for culture [46]. The secretome of SHEDs and young DPSCs contained more growth factors and lower levels of pro-inflammatory cytokines compared to DPSCs obtained from old subjects. Differentiation potential was also higher in SHEDs and young DPSCs [47].
CM can be obtained by healthy PDLSCs but also by inflamed PDLSCs. CM obtained by inflamed ones increased proliferation of both healthy and inflamed PDLSCs, but reduced the differentiation toward osteoblasts. Healthy CM rescued the impaired osteogenic differentiation [48].
The exposure to differentiation medium also could induce changes in non-coding RNA in EVs and EXOs of PDLSCs. Specifically, 69-557 circular RNA (circRNAs) and 2907-11,581 lncRNAs were found in EVs isolated from PDLSCs and PDLSCs exposed to osteogenic differentiation medium at different time points. Compared with undifferentiated PDLSCs EVs, 3 circRNAs and 2 lncRNAs were upregulated and 39 circRNAs and 5 lncRNAs were downregulated consistently after 5 and 7 days of exposure to differentiation medium [52]. Moreover, 72 miRNAs were upregulated while 35 were downregulated in PDLSCs EXOs after osteogenic induction [53].
A summary of the main factors found in the secretome of the different dental MSCs can be found in Table 1.  FGF-2modified GMSCs CM ↑ VEGF-A, FGF-2, TGF-β.

Dental Stem Cell Secretome Neuroprotective and Neuroregenerative Potential in Preclinical Models
In order to evaluate the neuroregenerative and neuroprotective potential of the dental MSC secretome, the effects of CM and EVs have been evaluated in preclinical models of neurodegenerative and neurological diseases and models of neuronal damage, such as spinal cord injury (SCI). In addition, secretome mediated effects on neuronal outgrowth, its capacity to stimulate neuronal differentiation, and the effects on glial cells have also been evaluated. We performed a PubMed search looking for studies showing the neuroregenerative and neuroprotective potential of dental MSCs secretome in in vitro and in vivo models.

Dental Pulp Stem Cell Secretome
DPSCs secretome was one of the most studied. Different studies evaluated its efficacy in inducing neurite outgrowth. It was reported that DPSC-CM promoted neurite outgrowth in dorsal root ganglion (DRG) neurons. Specifically, the total length and joint number of neurites increased after treatment with CM. Moreover, DPSC-CM promotes Schwann cell viability and myelin formation [54].
DPSCs-CM enhanced cell survival and induced neurite outgrowth of PC12 cells, as shown by neuronal nuclear protein (NeuN), microtubule-associated protein 2 (MAP-2) and βIII-tubulin. Specifically, DPSCs-CM was more efficacious in inducing PC12 neurite outgrowth compared to DPSCs/PC12 co-cultures, indicating that cells co-cultures had a delayed lag time in producing efficacious amounts of trophic factors. DPSCs-CM also enhanced cell migration. Interestingly, the number of surviving PC12 cells was reduced when CM was added with anti-GDNF. Instead, the addition of anti-NGF, anti-GDNF and anti-BDNF antibodies attenuate PC12 neurite outgrowth. These data demonstrated that NGF, BDNF and GDNF are involved in the PC12 survival and differentiation [55].
The DPSC secretome shows a chemoattractive effect on SH-SY5Y cells. Moreover, its effect on neural maturation has been evaluated. With this aim, SH-SY5Y cells were induced toward neuronal cells and after they were exposed to the DPSC secretome. SH-SY5Y cells subjected to the DPSC secretome showed increased neurite outgrowth, acquired ultrastructural features of neuronal cells and presented an increased immune reactivity for neuronal markers. Moreover, CM-treated SH-SY5Y cells developed distinct features including Cd2+-sensitive currents, which suggests that CM-DPSC-maturated SH-SY5Y acquired voltage-gated Ca 2+ channels [56]. In line with the previous study, CM obtained by DPSC sheet induced the formation and outgrowth of neurites in neuronally differentiated SH-SY5Y neuroblastoma cells. These effects were enhanced when DPSC sheets were cultured with FGF2. The neurite-promoting effects were abolished when neurotrophic factors were inhibited, suggesting that they are needed for the positive effect of DPSC sheets on neuronal cell activity [57].
Recently, Chouaib et al. evidenced that DPSC-CM enhancement of neurite outgrowth in sensory neurons is concentration dependent. The authors also found that 48 h of DPSCs conditioning was the best option to obtain CM with efficient activity, while extending the conditioning time did not improve the effects of DPSC-CM. Interestingly, the frozen storage did not influence experimental outcomes. The CM contained some factors known for their role in neurogenesis and neuroprotection but also in angiogenesis and osteogenesis. Moreover, the conditioning of DPSCs with the B-27 supplement enhanced the neuroregenerative effects of their secretome, inducing a change of its composition in growth factors. In particular, CM was more efficacious when B-27 was added to DPSCs before conditioning [58].
CM from DPSCs enhanced neuritogenesis and exerted a chemoattractant effect also on neural stem cells (NSCs). The priming of DPSCs with leukocyte-and platelet-rich fibrin (L-PRF) increased BDNF secretion, but exerted no additional effects on the paracrine-mediated repair mechanisms [59].
DPSC-derived CM was also shown to be able to protect and regenerate isolated primary trigeminal ganglion neuronal cells (TGNC). Indeed, CM enhanced TGNC survival associated with extensive neurite outgrowth and branching. In parallel, DPSC-CM significantly upregulated NeuN, βIII-tubulin, and synapsin-I neuronal marker expression as well as TRPV1. Interestingly, DPSC-CM contained NGF, BDNF, NT-3, and GDNF [60].
G-CSF-mobilized DPSCs expressed higher neurotrophic factors compared to basal DPSCs and their secretome showed an enhanced neurite extension potential. Indeed, mobilized DPSC CM had a greater effect on neurite outgrowth in TGW cells [61]. Previously, it was demonstrated that CM from mobilized DPSC enhanced proliferation and migratory activity of neuronal Schwann RT4-D6P2T cells [62].
Interestingly, CM from SHEDs and DPSCs was shown to be able to promote the regeneration of cerebral granular neurons inhibiting axon growth inhibitors signals by paracrine mechanisms [63].
The DPSCs secretome also shows superior effects compared to other MSCs. Kumar et al. demonstrated that the secretome derived from DPSCs, SCAPs and DFSCs induced neural differentiation in IMR-32 cells, a preneuroblastic cell line, in a more efficient manner compared to BMSCs. In particular, neurite length was higher when IMR-32 cells were treated with the DPSC secretome. The DPSC secretome contained GCSF, IFN-γ, and TGF-β, which may promote neural differentiation [64].
DPSCs, BMSCs and AMSCs promoted an increase in the survival of co-cultured retinal ganglion cells. In particular, the increase in survival was enhanced in DPSC-treated retinal cultures. Interestingly, coculture with DPSC induced a significant increase in both the number of neurite-bearing retinal ganglion cells and neurite length compared with cocultures with BMSCs and AMSCs. However, these effects were blocked using neurotrophic factor receptors Fc-receptor blockers. The different types of MSCs showed a different pattern of neurotrophic factor expression, and, specifically, DPSCs released higher levels of several growth factors such as NGF, BDNF, and VEGF compared with BMSCs and AMSCs. In particular, VGF may mediate the neuroprotective effects of DPSCs [65].
Venugopal et al. compared the neuroprotective potential of EXOs, CM or neuron-MSC-co-culture system against kainic-acid-induced excitotoxicity in vitro. Moreover, in order to identify the most adapt MSC type, EXOs and CM derived from DPSCs and BMSCs were tested. All three approaches showed neuroprotective potential thanks to the increase of growth factor expressions and the inhibition of apoptosis through the activation of PI3K-Bcl-2 pathway. It is important to note that EXOs demonstrated better anti-necrotic properties compared to neuron-MSC co-culture or CM. Regarding CM, only the fraction containing proteins in the range 3-10 kDa showed neuroprotection and rescued the neurons from excitotoxicity [67].
The secretome of DPSCs also showed beneficial effects in models of neurodegenerative diseases. Treatment with DPSC secretome reduced amyloid β (Aβ) cytotoxicity in an in vitro model of Alzheimer's disease (AD), increasing cell viability and reducing apoptosis. DPSC secretome was shown to contain elevated levels of VEGF, Fractalkine, RANTES, monocyte chemoattractant protein-1 (MCP-1), and GMCSF compared to those of BMSCs and AMSCs. Interestingly, neprilysin, a protease able to degrade Aβ, was also found in the DPSC secretome. DPSC secretome proteolytically degrades Aβ 1-42 in vitro, resulting in complete degradation after 12 h [68].
Early pre-symptomatic DPSC-CM administration improved neuromuscular junction innervation compared to vehicle-treated SOD1 G93A mice. The administration during late pre-symptomatic stages not only increased neuromuscular junction preservation, but also motor neuron survival in the spinal cord ventral horn. However, astrogliosis and microglia reactivity remained unaffected. Interestingly, the daily DPSC-CM treatment from symptom onset increased post-onset survival as well as overall lifespan [69].
DPSC-CM ameliorated aneurysmal subarachnoid hemorrhage (aSAH)-induced vasoconstriction and improved oxygenation in an injured brain. DPSC-CM administration also ameliorated cognitive and motor impairments. DPSC-CM administration decreased neuroinflammation as demonstrated by the reduction in the number of Iba1-positive cells. The major constituent of DPSC-CM was IGF-1. Antibody-mediated neutralization of IGF-1 moderately deteriorated the rescuing effect of DPSC-CM on microcirculation, Iba1-positive cells in the injured brain area, and the cognitive/motor impairments [71].
DPSC-CM administration ameliorated sciatic motor/sensory nerve conduction velocity, sciatic nerve blood flow, and intraepidermal nerve fiber density in the footpads of streptozotocin-induced diabetic rats. Furthermore, capillary density of the skeletal muscles increased while pro-inflammatory reactions in the sciatic nerves of diabetic rats were reduced [72]. Kanada et al. confirmed the positive effects of CM in sciatic nerve conduction velocity and sciatic nerve blood flow. Moreover, the treatment also increased muscle bundle size, vascular density in the skeletal muscles, and intraepidermal nerve fiber density in the diabetic rats. However, no differences were found between the results for DPSCs and DPSC-CM. These results suggested that the efficacy of DPSC and DPSC-CM administration were probably due to the secretome. In particular, DPSC-CM contained angiogenic factors such as VEGF-C, neurotrophic factors, such as BDNF, and immunomodulatory factors including IL-1β, IL-4, and TLR4 [73].

Stem Cells from Human Exfoliated Deciduous Teeth Secretome
Secretome of SHEDs was reported to modulate microglial cell activity. EVs derived from SHEDs inhibited lipopolysaccharide (LPS)-induced activation of NF-κB signaling pathway in human microglial cells. Moreover, EVs induced an upregulation of phagocytic activity in unpolarized cells, a slight decrease in M1 polarized cells, and a moderate increase in M2 polarized cells. EVs induced an immediate and sustained increase of glycolytic activity in M0, M1, and M2 polarized cells. Interestingly, EVs acted in an inverse dose-dependent manner [74]. EVs also induced a rapid increase in intracellular Ca 2+ and ATP release in microglial cells. EVs were also able to promote microglial motility through P2X4 receptor/milk fat globule-epidermal growth factor-factor VIII (MFG-E8)-dependent mechanisms [75].
Different studies report beneficial effects of SHED CM in both in vitro and in vivo Parkinson's disease (PD) models. Fujii et al. evidenced that dopaminergic-neuron-like cells induced from SHEDs were able to exert therapeutic benefits in a 6-hydroxy-dopamine (6-OHDA)-induced Parkinsonian rat model, improving neurological deficits and increasing dopamine (DA) levels more efficiently than undifferentiated SHEDs. However, paracrine effects may contribute to neuroprotection against 6-OHDA-induced neurodegeneration. Indeed, the CM obtained from differentiated SHEDs was able to protect primary neurons against 6-OHDA toxicity and accelerated neurite outgrowth in vitro [76]. Different doses of SHED-CM were tested in a PD model. The dose 10 µg/mL of SHED-CM did not restore motor ability, while 30 µg/mL of SHED-CM induced only mild improvements. Instead, 100 µg/mL of SHED-CM induced the maximal improvement of motor deficits in PD rats and a higher dose did not induce further improvement. SHED-CM increased tyrosine hydroxylase (TH) amounts and decreased synuclein levels in both the substantia nigra and striatum. In addition, SHED-CM treatment decreased both Iba-1 positive cells and CD4 levels in the same brain areas. The major constituents of SHED-CM included insulin-like growth factor binding protein-6 (IGFBP-6), tissue inhibitor of metalloproteinase (TIMP)-2, TIMP-1, and TGF-1. Moreover, bioinformatics analysis indicated that SHED-CM was able to promote neural regeneration. Indeed, RNA sequencing evidenced that SHED-CM administration shifted the gene expression profile to a pattern similar to that of control rats, upregulating genes that were involved in neurodevelopment and nerve regeneration. The major constituents of SHED-CM may participate in the molecular networks involved in cholinergic and serotoninergic synapses, calcium signaling pathways, and axon guidance [77].
EXOs and MVs derived from SHEDs have also been evaluated for their neuroprotective effects in PD models. EXOs, but not MVs, derived from SHEDs grown on laminincoated three-dimensional alginate micro-carriers suppressed 6-OHDA-induced apoptosis in dopaminergic neurons. On the contrary, no protective effects were exerted by MVs or EXOs derived from SHEDs grown in standard culture conditions [78].
Instead, intranasal administration of EVs derived from SHEDs was shown to be effective in a rat model of PD, improving motor function in association with a normalization of TH expression in the striatum and substantia nigra [79].
The intranasal administration was also tested in an AD model, showing that SHED-CM improved cognitive function. SHED-CM reduced oxidative stress, shifted the M1-type pro-inflammatory microenvironment toward the M2-type anti-inflammatory and neuroprotective one, and increased neurotrophic factor levels. BMSCs-CM was less efficacious. It reduced oxidative stress and inflammation, but could not upregulate the expression of the anti-inflammatory M2 markers. Treatment with SHED-CM also suppressed glutamateinduced neuronal death in vitro [80]. SHED-CM was also able to improve disease scores and reduce demyelination, axonal injury, inflammatory cell infiltration, and proinflammatory cytokine expression in the spinal cord of experimental autoimmune encephalomyelitis (EAE) mice. These changes were associated with a change in the microglia/macrophage phenotype from M1 to M2. The treatment of EAE mice with the secreted ectodomain of sialic-acid-binding Ig-like lectin-9 (ED-Siglec-9), a major component of SHED-CM, resulted in similar effects compared to SHED-CM treatment, while ED-Siglec-9 depletion abolished the protective effects of SHED-CM. On the contrary, HGF depletion did not cause an inhibition of SHED-CM mediated protection, indicating that HGF had little effect on the efficacy of SHED-CM. SHED-CM inhibited the proliferation of myelin oligodendrocyte glycoprotein-specific CD4+ T cells, as well as their production of proinflammatory cytokines in vitro [81]. Matsubara et al. showed that SHED and SHED-CM administered into rat injured spinal cord during the acute postinjury period induced functional recovery. SHED-CM showed anti-inflammatory activity, reducing the levels of pro-inflammatory cytokines, and immunoregulatory action, inducing M2 anti-inflammatory macrophages. To identify factors responsible for the therapeutic effects of CMs, soluble factors present in SHED-CM were characterized. A total of 79 proteins were identified, some of them known to be involved in neuroregenerative processes, with anti-apoptotic, anti-inflammatory and axonal elongation properties. In particular, MCP-1 and ED-Siglec-9 may be involved in M2-like macrophage differentiation. Indeed, depleting these factors from the SHED-CM reduced CM's ability to induce M2-like macrophages and to promote functional recovery after SCI. Interestingly, the administration of BMSC-CM induced no or only slight M2-like cell differentiation and did not induce recovery such as SHED-CM [82].
In agreement with the previous study, the treatment with SHED-CM loaded on a collagen hydrogel, used as a delivery system, induced functional recovery in SCI rats, as demonstrated by improvement in scores evaluated through Basso, Beattie, and Bresnahan scoring, inclined plane, cold allodynia, and beam walk tests [83]. The treatment with SHED-CM loaded on a collagen hydrogel also increased the volume of preserved white and gray matter and the total number of neurons and oligodendrocytes in a rat SCI model. On the contrary, lesion volume and lesion length decreased. However, in this study SHED-CM alone exerted no protection. The authors suggested that this may be due to the rapid diffusion of SHED-CM, and thus collagen hydrogel may act as an efficient releasing system [84].
A single intravenous injection of SHED-CM also reversed the mechanical allodynia induced by spinal nerve transection, suppressed microglia and astrocytes activation, and decreased the numbers of neurons positive for the neuronal injury marker activating transcription factor 3 (ATF3) and macrophage accumulation. In particular, the SHED-CM fraction with a molecular weight between 30 and 50 kDa reversed the pain, suggesting that protein components with molecular mass in the range 30-50 kDa were responsible for the reported neuroprotection [85].
The implantation of a collagen sponge enriched with the serum-free CM from SHED into the nerve gap formed by rat facial nerves transection restored the neurological function. On the contrary, CM depleted of MCP-1 and ED-Siglec-9, which are anti-inflammatory M2 macrophage inducers, did not restore neurological function. Notably, MCP-1 and ED-Siglec-9 induced the polarization of M2 macrophages in vitro and in vivo. Thus, the results indicated that MCP-1/ED-Siglec-9 participated in peripheral nerve regeneration inducing M2 macrophage [86].
SHED-CM treatment increased proliferation, migration, and the expression of neuron-, ECM-, and angiogenesis-related genes in Schwann cells. Moreover, SHED-CM stimulated neurite outgrowth of dorsal root ganglia and increased cell viability. In vivo, axon regeneration and myelination were higher in the SHED-CM group after nerve transection surgery. Motor function improved while muscle atrophy was reduced in the SHED-CM group. Thus, SHEDs may secrete various trophic factors that enhance peripheral nerve regeneration through multiple mechanisms. Specifically, SHED-CM contained NGF, BDNF, NT-3, GDNF, ciliary neurotrophic factor (CNTF), VEGF, and HGF [87].
The administration of SHED-EXOs improved rat motor functional recovery and reduced cortical lesion in rats with traumatic brain injury. SHED-EXOs can exert these effects, reducing neuroinflammation shifting microglia polarization [88].
SHED-CM induced an improvement of motor disability and reduced infarct volume after permanent MCAO. The SHED-CM treated group showed increased levels of doublecortin, neurofilament H, neuronal nuclei and rat endothelial cell antigen in the peri-infarct area. Interestingly, SHED-CM induced the migration and differentiation of endogenous neuronal progenitor cells (NPC), vasculogenesis and improved ischemic brain injury [89].
Intracerebral administration of SHED-CM in hypoxia-ischemia injured mice improved neurological function, survival rate, and neuropathological score [90].
CM obtained from SHEDs, and specifically only the fraction of <6 kDa, promoted neurite outgrowth of DRG neurons. Moreover, SHED-CM prevented the decline in sensory nerve conduction velocities in diabetic mice and ameliorated the capillary number-tomuscle fiber ratio and capillary blood flow [91].
In an animal model of superior laryngeal nerve injury, the systemic administration of SHED-CM induced functional recovery, increasing the degree of myelination and promoted axonal regeneration shifting macrophages toward the M2 phenotype [92].

Periodontal Ligament Stem Cell Secretome
CM obtained from PDLSCs of relapsing remitting multiple sclerosis (RR-MS) patients showed anti-inflammatory and antiapoptotic effects in NSC-34 mouse motor neurons stimulated with the medium of LPS treated RAW 264.7 macrophages. Indeed, CM treatment reduced TLR4 and NF-κB levels, together with pro-inflammatory cytokines. On the contrary, IL-10 increased in association with neuroprotective markers such as nestin, neurofilament 70, NGF, and GAP43. Interestingly, the neuroprotective effects of EVs may be due to their content in IL-10 and TGF-β [93].
The effects of PDLSCs-CM obtained from healthy donors and RR-MS patients were also evaluated in phorbol-12-myristate-13-acetate (PMA) differentiated THP-1, used as a model of microglia, and in undifferentiated and differentiated MO3.13 cells, used as models of progenitor cells and oligodendrocytes, respectively, treated with Porphyromonas gingivalis LPS. Treatment with both CM reduced the LPS-induced increase in TNFα, IL-1β and IL-6 levels and reduced TLR-4 in THP-1 cells [94].
PDLSCs-CM and purified EXOs/MVs (PDLSCs-EMVs) obtained from RR-MS patients and healthy donors exerted protective effects in EAE mice. In particular, PDLSCs-CM and PDLSCs-EMVs improved disease scores, restoring tissue integrity and remyelination in the spinal cord. PDLSCs-CM and PDLSCs-EMVs exerted anti-inflammatory effects both in spinal cord and spleen, as demonstrated by the reduction of pro-inflammatory cytokines and the induction of IL-10. In parallel, apoptosis was also inhibited. The anti-inflammatory effect of CM or EMVs might be due to the presence of the immunomodulatory cytokines IL-10 and TGF-β [95]. Moreover, PDLSCs-CM and EMVs obtained from RR-MS patients inhibited NALP3 inflammasome activation and reduced TLR-4 and NF-κB levels in EAE mice. The immunomodulatory factors IL-10, TGF-β, and SDF-1α included in the CM may be responsible for the immunosuppressive role of PDLSCs-CM and EMVs in EAE [96].
Interestingly, CM obtained from PDLSCs cultured under hypoxic conditions was efficacious in ameliorating clinical and histologic disease scores in EAE mice. In particular, this treatment reduced inflammatory cell infiltration and increased remyelination in the spinal cord. In particular, hypoxic CM administration increased the anti-inflammatory cytokine IL-37 in association with the reduction of proinflammatory cytokines. Moreover, oxidative stress and apoptosis were also inhibited, while BDNF increased. CM treatment was also able to regulate autophagy through the activation of the PI3K/Akt/mTOR pathway. Furthermore, in in vitro scratch injury exposed NSC-34 motor neurons, the hypoxia CM was able to modulate inflammation, oxidative stress, and apoptosis. Interestingly, hypoxia CM contained NT-3, IL-10, and TGF-β that may explain its neuroprotective effects [97].
An overview of the studies presented in this paragraph is available in Table 4.

Other Dental-Derived MSCs
Neuroprotective effects of CM obtained by GMSCs were evaluated in mechanically injured murine motor-neuron-like NSC-34 cells. CM treatment reduced the scratch injuryinduced apoptosis and oxidative stress. Moreover, CM reduced TNF-α while increasing the levels of the anti-inflammatory cytokine IL-10. Interestingly, CM treatment upregulated BDNF and NT-3. CM was shown to contain NGF, NT-3, IL-10, and TGF-β, which may explain the neuroprotective effects [98].
EVs from GMSCs were tested for peripheral nerve regeneration in a crush-injured sciatic nerve mouse model. In vivo, the transplantation of Gelfoam embedded with GMSCderived EVs at the crush injury site induced functional recovery and axonal regeneration in a similar way compared to the direct transplantation of GMSCs. In particular, EVs promoted proliferation and migration of Schwann cells and upregulated the protein expressions of c-JUN, Notch1, GFAP, and SOX2 genes associated with dedifferentiation or repair phenotype of Schwann cells. Also in vitro, EVs promoted the expression of Schwann cell dedifferentiation/repair genes [99]. A positive effect on Schwann cell proliferation was also reported for EXOs from GMSCs, which also promoted DRG axon growth in vitro. Moreover, the effects of GMSCs EXOs combined with biodegradable chitin conduits on peripheral nerve regeneration were evaluated. In vivo, in a rat sciatic nerve defect model, chitin conduit combined with EXOs increased the number and diameter of nerve fibers and promoted myelin formation. In parallel, nerve conduction also improved. Moreover, muscle function and motor function were ameliorated [100].
CM from SCAPs, DPSCs, and PDLSCs were tested to evaluate their capacity in inducing neurite outgrowth. With this aim, differentiated neuroblastoma SH-SY5Y cells were incubated with the different CM. The CM were shown to be able to increase the percentage of cells producing neurites and the total neurite outgrowth length. Interestingly, the length of the longest neurite per neuron was increased in a significant manner only with SCAP CM, and the neutralization of the secreted BDNF inhibited neurite outgrowth, indicating its importance in this process [101]. CM released from SCAPs showed also a greater neurogenic inductive effect on DPSCs compared to BMSCs-CM. Indeed, when DPSCs were cultured in medium for neural stem cell growth, the levels of neurogenic markers increased with the addition of SCAPs-CM. On the contrary, neuronal marker expression was reduced, while that of neurotrophic marker increased, when BMSCs-CM was added. Cell proliferation was not influenced by SCAPs-CM [102].
Oral mucosa stem cells (OMSCs) were differentiated into cells showing an astrocytelike morphology and expressed characteristic astrocyte markers. The CM obtained by differentiated OMSCs increased the cell viability of motor neurons cultured in hypoxic conditions or exposed to hydrogen peroxide in vitro [103].
An overview of the studies presented in this paragraph is available in Table 5.

Translation of Secretome Application from the Preclinical Models into Clinical Use
The application of the secretome for a cell-free therapy may present some advantages compared to the use of MSCs. The main advantages of the use of the secretome instead of stem cell therapy are represented by the low immunogenicity and easier production, handling, and storage of the secretome [104]. Then, this therapy can overcome the risks linked to cell therapy, such as tumorgenicity, antigenicity, host rejection, and infections. Secretome handling can be easier compared to cells, given that it can be concentrated, frozen, and that it does not require liquid nitrogen storage and cell culture facilities, making its transfer also easier [105,106]. Moreover, secretome production is more economical and mass production under controlled laboratory conditions is also possible [107]. However, the period of survival of MSCs after transplantation is not clear, given that some data seem to indicate a limited survival [23].
Thus, therapeutic strategies based on secretome application could be helpful in regenerative medicine, based on their content in bioactive molecules, including proteins, mRNAs but also non-coding RNA, that can be useful to induce repair mechanism in the injured tissues. However, before translation into clinical applications, several points need to be clarified. In particular, it is necessary to better define the secretome composition, dosage, frequency, and route of administration. In this regard, it is also necessary to develop standardized manufacturing protocols with good manufacturing practice for the development of new pharmaceuticals based on cell-free products [105]. Indeed, for applications into clinical practice, the secretome should be presented in a standardized and easy to handle formulation. The secretome indeed may present variations on the basis of the subjects, cells, and tissues of origin [30]. For this reason, to standardize the secretome production it is necessary to define culture medium and supplements, culture duration, and culture conditions [106]. In this context, big data studies that evaluate proteome, transcriptome and non-coding RNA profile may provide assistance for secretome characterization. A list of studies analyzing transcriptome, non-coding RNA, and proteome profiling is available in Table 6. Table 6. Studies analyzing proteome, transcriptome, and non-coding RNA profiles of different dental MSCs.

MSCs Type
Secretome Profile Ref.
However, secretome delivery also needs to address the concern of the rapid diffusion and clearance of secretome from the tissue repair site. Moreover, secretome stability and the stability of its growth factors and miRNAs need to be maintained in physiological conditions for all the delivery duration. In this context, different biomaterials have been developed and optimized in order to improve the delivery efficiency of MSCs secretome, with the advantages of a prolonged release duration, protection against degradation, and enhancing the therapeutic capacity [108]. Another option to improve the long-lasting stability of formulations is a freeze-drying process, which is used for different biological products [109,110].

Conclusions
Dental MSCs, given their origin from neural crest, have been shown to possess a prominent neuroregenerative potential. Dental MSC-derived secretome also shows the same enhanced neuroprotective and neuroregenerative properties. Both CM and EVs contain neurotrophins and molecules with a neuroprotective action, even at higher levels compared to other MSCs. The studies evaluated in this review highlighted that both CM and EVs stimulated neurite outgrowth and showed neuroprotective effects in preclinical models of neurological diseases and neuronal damage. In particular, the secretomes of DPSCs and SHEDs were the most studied, but different studies also highlighted the neuroprotective effects of PDLSC and GMSC secretomes. Interestingly, some studies also suggested the superiority of the secretome obtained from dental MSCs compared to other MSCs sources, such as BMSCs and AMSCs, for neuroprotection. In conclusion, the secretome derived from dental MSCs seems promising for its application in the neuroregenerative field and may be useful to develop new neuroprotective treatments.

Conflicts of Interest:
The authors declare no conflict of interest. The funder had no role in the design of the study; in the collection, analyses, or interpretation of data; in the writing of the manuscript, or in the decision to publish the results.